There have been increasing demands for rapid and/or easy to operate assays for detecting the presence of analytes in liquid samples in fields such as clinical and forensic medicine, environmental resting, food contamination testing, and drug use testing. Particularly in demand are rapid, preferably single-step, assays that detect specific an analyte and can be performed outside of the laboratory setting, such as in homes, doctor's offices, or remote locations. A growing number of studies have been focused on analyte detection devices that can perform such rapid assays in detecting a given analyte in biological samples.
Typical of such rapid analyte detection assays and devices are the so called “dipstick,” lateral flow,” and “flow through” format of assays and devices. The dipstick format of assays and devices are exemplified in U.S. Pat. Nos. 4,059,407; 5,275,785; 5,504,013; 5,602,040; 5,622,871; and 5,656,503, the contents of which are incorporated herein by reference. A typical dipstick device consists of a strip of porous material having a sample receiving end, a reagent zone, and a reaction zone. It may also contain an absorbent material to the end of the reaction zone to absorb the excess liquid. Different materials, usually porous, may be used for the sample receiving zone, reagent zone, and reaction zone. The materials may be combined to form a single strip.
When using a dipstick device for analyte detection, a liquid sample is first applied to the sample receiving zone or the sample receiving zone is dipped into the liquid sample. The liquid sample is then wicked along the strip toward the reagent zone where the analyte binds to a reagent, which has been pre-incorporated into the strip at the reagent zone, to form a complex. Typically, the complex is an antibody/antigen complex or a receptor/ligand complex having a label. The labeled complex then migrates into the reaction zone where the complex binds to another specific binding partner, which is firmly immobilized in the reaction zone, resulting in a visible readout. The remaining liquid may then be absorbed into the absorbant material.
Typical lateral flow devices utilize a porous material that has a linear construction similar to that of the lipstick device: a sample reception zone, a reagent releasing zone, and a reaction zone. However, instead of vertically wicking the sample up the dipstick, lateral flow devices allow the sample to flow laterally across the porous material. Examples of assays and devices using the lateral flow format can be found in U.S. Pat. Nos. 5,075,078; 5,096,837; 5,229,073; 5,354,692; 6,316;205; and 6,368,876, the contents of which are incorporated herein by reference.
During an assay using the lateral flow format, a liquid sample containing the analyte is applied onto the sample receiving area. The sample is then transported through the sample receiving area, usually via capillary action, to the reagent area, which is sometimes called the “conjugate release area;” or to the reaction area, which is sometimes called the “analytical membrane,” depending on the test and device is configured. The reagent area is usually impregnated or striped with a reversibly bound conjugate, as well as optional buffer, surfactant, and/or protein. When the sample travels into the reagent area, the analyte binds to the conjugate and the analyte/conjugate complex is re-suspended. The liquid sample may also solubilize the optional additives such as surfactant, detergent and protein that help with the overall flow. When the analyte/conjugate complex travels to the reaction area or the analytical membrane, the analyte binds with an immobilized and usually labeled secondary reactant (e.g., an antibody such as an enzyme labeled with colored latex particles or colloid). The presence of the analyte is thus visually detected. The analytical membrane may contain two distinct regions, a test region and a control region, also know as the end of assay indicator. An absorbant material may be used to control the flow through the device by pulling excess reagents from the reaction area. The absorbant material is also important in diminishing assay background.
A flow-through device, in some instances, contains components analogous to those used in a lateral flow device. The components in such a flow through device, however, are stacked one on top of the other for a unilateral downward flow through. Typically in such a flow through device, the sample application pad lies on top of and in direct contact the conjugate pad, which in turn lies atop the analytical membrane, which lies above the absorbant pad.
A flow-through device may contain only a porous membrane and, optionally, a housing and an absorbant material. This type of flow-through device is disclosed in U.S. Pat. No. 4,632,901, the content of which is incorporated herein by reference. In typical assays using such a flow-through device, a liquid sample is applied to the porous membrane, on which a reagent, such as an antibody, has been bound. If the analyte, such as an antigen, is present in the liquid sample, the analyte will be bound to the antibody. Then, another solution of a labeled reagent, such as a labeled antibody, is added to the porous membrane. A washing step usually follows to remove unbound labeled antibody. The labeled reagent, indicates the presence of the analyte.
As can be seen from the above description of typical analyte detection devices, the sample receiving area, reagent area, reaction area or analytical membrane, and the absorbant material may be all made from porous materials, such as porous polymeric materials. As also can be seen, a key component in such devices is the porous membrane material wherein the reaction between the analyte, such as an antigen, and the reagent, such as an antibody, occurs. The porous membrane is usually called the analytical membrane, reaction membrane, or membrane. Thus, the analytical membrane can be the porous strip in a dipstick device, the reagent zone or analytical membrane in a lateral flow device, or the analytical membrane in a flow through device. The membrane performs the critical part of facilitating the reaction between the analyte and reagent and their detection thereafter.
As also can be seen, three properties of a porous material are important to its use as the membrane of an analyte detection system: reagent (e.g., protein) binding ability, porosity, and strength. The ability of the membrane to immobilize reagents, such as proteins and other macromolecules, is paramount because, together, they form the solid phase used in the assay. The porosity of the membrane is important because reactants must be able to flow through the matrix so that the membrane can separate bound from free components. The strength of the membrane is important for the design, manufacture, and use of the device.
Nitrocellulose, Nylon, poly(vinylidene fluoride) (PVDF), and polysulfones (PS) materials have been used as the membrane in analyte detection devices. Nitrocellulose is currently the material of choice because of its versatility and sensibility that makes it avoid high level of nonspecific interactions. However, because of certain requirements, such as a web membrane casting process, in nitrocellulose manufacturing process, membranes made from nitrocellulose may have inconsistent properties in binding ability, porosity, and/or strength. Designers of analyte detection devices sometimes may need to re-optimize assay conditions for nitrocellulose materials from different production lots.
Nylon membranes, while generally having higher protein binding abilities than nitrocellulose membranes, tend to result in higher levels of nonspecific bindings, making their applications limited in certain areas. PVDF and PS membranes are capable of producing consistent assay results. However, due to factors such as cost of production, their application in analyte detection devices has not been widely accepted.
It is clear that there is a need for materials that can be used as membranes in an analyte detection device and, at the same time, avoid one or more of the drawbacks discussed above. More specifically, there is a need for porous polymeric materials useful as membranes in analyte detection devices that can be produced economically and consistently. A need also exists for materials that have strong and specific binding abilities for a wide range of reagents, such as proteins and other macromolecules, porosities that can be accurately controlled, and strengths that make their integration into devices easy and flexible.